This invention relates to DNA constructs, replicable expression vectors containing the constructs, bacteria containing the constructs and vaccines containing the bacteria or fusion proteins expressed therefrom. More particularly, the invention relates to novel DNA constructs encoding the C-fragment of tetanus toxin, and to fusion proteins containing tetanus toxin C-fragment.
It is known to prepare DNA constructs encoding two or more heterologous proteins with a view to expressing the proteins in a suitable host as a single fusion protein. However, it has often been found that fusing two proteins together in this way leads to an incorrectly folded chimaeric protein which no longer retains the properties of the individual components. For example, the B-subunits of the Vibrio cholerae (CT-B) and E. coli (LT-B) enterotoxins are powerful mucosal immunogens but genetic fusions to these subunits can alter the structure and properties of the carriers and hence their immunogenicity (see M. Sandkvist et al. J. Bacteriol. 169, pp4570-6, 1987, Clements et al. 1990 and M. Lipscombe et al. Mol. Microbiol. 5, pp 1385, 1990). Moreover, many heterologous proteins expressed in bacteria are not produced in soluble properly folded or active forms and tend to accumulate as insoluble aggregates (see C. Schein et al. Bio/Technology 6, pp 291-4, 1988 and R. Halenbeck et al. Bio/Technology 7, pp 710-5, 1989.
In our earlier unpublished international patent application PCT/GB93/01617, it is disclosed that by providing a DNA sequence encoding tetanus toxin C-fragment (TetC) linked via a xe2x80x9chinge regionxe2x80x9d to a second sequence encoding an antigen, the expression of the sequence in bacterial cells is enhanced relative to constructs wherein the C-fragment is absent. For example, the expression level of the full length P28 glutathione S-tranferase protein of S. mansoni when expressed as a fusion to TetC from the nirB promoter was greater than when the P28 protein was expressed alone from the nirB promoter. The TetC fusion to the full length P28 protein of S. mansoni was soluble and expressed in both E. coli and S. typhimurium. In addition, the TetC-P28 fusion protein was capable of being affinity purified by a glutathione agarose matrix, suggesting that the P28 had folded correctly to adopt a conformation still capable of binding to its natural substrate. It was previously considered that a hinge region, which typically is a sequence encoding a high proportion of proline and/or glycine amino acids, is essential for promoting the independent folding of both the TetC and the antigenic protein fused thereto. However, it has now been discovered, surprisingly in view of the previous studies on CT-B and LT-B referred to above, that when the hinge region is omitted between the TetC and a second antigen such as P28, the proteins making up the fusion do exhibit correct folding as evidenced by affinity purification on a glutathione agarose matrix.
Accordingly, in a first aspect, the invention provides a DNA construct comprising a DNA sequence encoding a fusion protein of the formula TetC-(Z)a-Het, wherein TetC is the C fragment of tetanus toxin, or a protein comprising the epitopes thereof; Het is a heterologous protein; Z is an amino acid, and a is zero or a positive integer, provided that (Z)a does not include the sequence Gly-Pro.
Typically (Z)a is a chain of 0 to 15 amino acids, for example 0 to 10, preferably less than 6 and more preferably less than 4 amino acids.
In one embodiment (Z)a is a chain of two or three amino acids, the DNA sequence for which defines a restriction endonuclease cleavage site.
In another embodiment, a is zero.
Usually the group (Z)a will not contain, simultaneously, both glycine and proline, and generally will not contain either glycine or proline at all.
In a further embodiment, (Z)a is a chain of amino acids provided that when a is 6 or more, (Z)a does not contain glycine or proline.
The group (Z)a may be a chain of amino acids substantially devoid of biological activity.
In a second aspect the invention provides a replicable expression vector, for example suitable for use in bacteria, containing a DNA construct as hereinbefore defined.
In another aspect, the invention provides a host (e.g. a bacterium) containing a DNA construct as hereinbefore defined, the DNA construct being present in the host either in the form of a replicable expression vector such as a plasmid, or being present as part of the host chromosome, or both.
In a further aspect, the invention provides a fusion protein of the form TetC-(Z)a-Het as hereinbefore defined, preferably in substantially pure form, said fusion protein being expressible by a replicable expression vector as hereinbefore defined.
In a further aspect the invention provides a process for the preparation of a bacterium (preferably an attenuated bacterium) which process comprises transforming a bacterium (e.g. an attenuated bacterium) with a DNA construct as hereinbefore defined.
The invention also provides a vaccine composition comprising an attenuated bacterium, or a fusion protein, as hereinbefore defined, and a pharmaceutically acceptable carrier.
The heterologous protein xe2x80x9cHetxe2x80x9d may for example be a heterologous antigenic sequence, e.g. an antigenic sequence derived from a virus, bacterium, fungus, yeast or parasite.
Examples of viral antigenic sequences are sequences derived from a type of human immunodeficiency virus (HIV) such as HIV-1 or HIV-2, the CD4 receptor binding site from HIV, for example from HIV-1 or -2., hepatitis A, B or C virus, human rhinovirus such as type 2 or type 14, Herpes simplex virus, poliovirus type 2 or 3, foot-and-mouth disease virus (FMDV), rabies virus, rotavirus, influenza virus, coxsackie virus, human papilloma virus (HPV), for example the type 16 papilloma virus, the E7 protein thereof, and fragments containing the E7 protein or its epitopes; and simian immunodeficiency virus (SIV).
Examples of antigens derived from bacteria are those derived from Bordetella pertussis (e.g. P69 protein and filamentous haemagglutinin (FHA) antigens), Vibrio cholerae, Bacillus anthracis, and E.coli antigens such as E.coli heat Labile toxin B subunit (LT-B), E.coli K88 antigens, and enterotoxigenic E.coli antigens. Other examples of antigens include the cell surface antigen CD4, Schistosoma mansoni P28 glutathione S-transferase antigens (P28 antigens) and antigens of flukes, mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and malaria parasites, eg. parasites of the genus plasmodium or babesia, for example Plasmodium falciparum, and peptides encoding immunogenic epitopes from the aforementioned antigens.
Particular antigens include the full length Schistosoma mansoni P28, and oligomers (e.g. 2, 4 and 8-mers) of the immunogenic P28 aa 115-131 peptide (which contains both a B and T cell epitope), and human papilloma virus E7 protein, Herpes simplex antigens, foot and mouth disease virus antigens and simian immunodeficiency virus antigens.
The DNA constructs of the present invention may contain a promoter whose activity is induced in response to a change in the surrounding environment. An example of such a promoter sequence is one which has activity which is induced by anaerobic conditions. A particular example of such a promoter sequence is the nirB promoter which has been described, for example in International Patent Application PCT/GB92/00387. The nirB promoter has been isolated from E.coli, where it directs expression of an operon which includes the nitrite reductase gene nirB (Jayaraman et al, J. Mol. Biol. 196, 781-788, 1987), and nirD, nirC, cysG (Peakman et al, Eur. J. Biochem. 191, 315323, 1990). It is regulated both by nitrite and by changes in the oxygen tension of the environment, becoming active when deprived of oxygen, (Cole, Biochem, Biophys. Acta. 162, 356-368, 1968). Response to anaerobiosis is mediated through the protein FNR, acting as a transcriptional activator, in a mechanism common to many anaerobic respiratory genes. By deletion and mutational analysis the part of the promoter which responds solely to anaerobiosis has been isolated and by comparison with other anaerobically regulated promoters a consensus FNR-binding site has been identified (Bell et al, Nucl, Acids. Res. 17, 3865-3874, 1989; Jayaraman et al, Nucl, Acids, Res. 17, 135-145, 1989). It has also been shown that the distance between the putative FNR-binding site and the xe2x88x9210 homology region is critical (Bell et al, Molec. Microbiol.4, 1753-1763, 1990). It is therefore preferred to use only that part of the nirB promoter which responds solely to anaerobiosis. As used herein, references to the nirB promoter refer to the promoter itself or a part or derivative thereof which is capable of promoting expression of a coding sequence under anaerobic conditions. The preferred sequence, and which contains the nirB promoter is:
AATTCAGGTAAATTTGATGTACATCAAATGGTACCCCTTGCTGAATCGTTAAGG TAGGCGGTAGGGCC (SEQ ID NO: 1)
In a most preferred aspect, the present invention provides a DNA molecule comprising the nirB promoter operably linked to a DNA sequence encoding a fusion protein as hereinbefore defined.
In another preferred aspect of the invention, there is provided a replicable expression vector, suitable for use in bacteria, containing the nirB promoter sequence operably linked to a DNA sequence encoding a fusion protein as hereinbefore defined.
The DNA molecule or construct may be integrated into the bacterial chromosome, e.g. by methods known per se, and thus in a further aspect, the invention provides a bacterium having in its chromosome, a DNA sequence or construct as hereinbefore defined.
Stable expression of the fusion protein can be obtained in vivo. The fusion protein can be expressed in an attenuated bacterium which can thus be used as a vaccine.
The attenuated bacterium may be selected from the genera Salmonella, Bordetella, Vibrio, Haemophilus, Neisseria and Yersinia. Alternatively, the attenuated bacterium may be an attenuated strain of enterotoxigenic Escherichia coli. In particular the following species can be mentioned: S.typhixe2x80x94the cause of human typhoid; S.typhimuriumxe2x80x94the cause of salmonellosis in several animal species; S.enteritidisxe2x80x94a cause of food poisoning in humans; S.choleraesuisxe2x80x94a cause of salmonellosis in pigs; Bordetella pertussisxe2x80x94the cause of whooping cough; HaemoDhilus influenzaexe2x80x94a cause of meningitis; Neisseria gonorrhoea the cause of gonorrhoea; and Yersiniaxe2x80x94a cause of food poisoning.
Examples of attenuated bacteria are disclosed in, for example EP-A-0322237 and EP-A-0400958, the disclosures in which are incorporated by reference herein.
An attenuated bacterium containing a DNA construct according to the invention, either present in the bacterial chromosome, or in plasmid form, or both, can be used as a vaccine. Fusion proteins (preferably in substantially pure form) expressed by the bacteria can also be used in the preparation of vaccines. For example, a purified TetC-P28 fusion protein in which the TetC protein is linked via its C-terminus to the P28 protein with no intervening hinge region has been found to be immunogenic on its own. In a further aspect therefore, the invention provides a vaccine composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, an attenuated bacterium or fusion protein as hereinbefore defined.
The vaccine may comprise one or more suitable adjuvants.
The vaccine is advantageously presented in a lyophilised form, for example in a capsular form, for oral administration to a patient. Such capsules may be provided with an enteric coating comprising, for example, Eudragit xe2x80x9cSxe2x80x9d, Eudragit xe2x80x9cLxe2x80x9d, Cellulose acetate, Cellulose acetate phthalate or Hydroxypropylmethyl Cellulose. These capsules may be used as such, or alternatively, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is advantageously effected in buffer at a suitable pH to ensure the viability of the organisms. In order to protect the attenuated bacteria and the vaccine from gastric acidity, a sodium bicarbonate preparation is advantageously administered before each administration of the vaccine. Alternatively, the vaccine may be prepared for parenteral administration, intranasal administration or intramammary administration.
The attenuated bacterium containing the DNA construct or fusion protein of the invention may be used in the prophylactic treatment of a host, particularly a human host but also possibly an animal host. An infection caused by a microorganism, especially a pathogen, may therefore be prevented by administering an effective dose of an attenuated bacterium according to the invention. The bacterium then expresses the fusion protein which is capable of raising antibody to the micro-organism. The dosage employed will be dependent on various factors including the size and weight of the host, the type of vaccine formulated and the nature of the fusion protein.
An attenuated bacterium according to the present invention may be prepared by transforming an attenuated bacterium with a DNA construct as hereinbefore defined. Any suitable transformation technique may be employed, such as electroporation. In this way, an attenuated bacterium capable of expressing a protein or proteins heterologous to the bacterium may be obtained. A culture of the attenuated bacterium may be grown under aerobic conditions. A sufficient amount of the bacterium is thus prepared for formulation as a vaccine, with minimal expression of the fusion protein occurring.
The DNA construct may be a replicable expression vector comprising the nirB promoter operably linked to a DNA sequence encoding the fusion protein. The nirB promoter may be inserted in an expression vector, which already incorporates a gene encoding one of the heterologous proteins (e.g. the tetanus toxin C fragment), in place of the existing promoter controlling expression of the protein. The gene encoding the other heterologous protein (e.g. an antigenic sequence) may then be inserted. The expression vector should, of course, be compatible with the attenuated bacterium into which the vector is to be inserted.
The expression vector is provided with appropriate transcriptional and translational control elements including, besides the nirB promoter, a transcriptional termination site and translational start and stop codons. An appropriate ribosome binding site is provided. The vector typically comprises an origin of replication and, if desired, a selectable marker gene such as an antibiotic resistance gene. The vector may be a plasmid.